ABSTRACT The goal of this project is to perform high-content analysis of drug and immunotherapy responses on hundreds of intact, live cultured fragments isolated from a single live tumor biopsy. In recent years, patient-derived tumor ?organoids? have shown great promise to predict drug responses for personalized cancer treatment. Immunotherapy, including cellular immunotherapy, represents the next generation of cancer therapy, and many of the relevant drugs act on the local tumor microenvironment (TME). There is a pressing need for functional testing platforms that use human, intact and live tumor tissue to better predict traditional and immunotherapy responses. Such platforms should also retain as much of the native TME as possible. Present high-throughput testing platforms that have some of these features, e.g. based on patient-derived tumor organoids, require a growth step that alters the TME. On the other hand, the micro-dissection of tumor tissue into ?spheroids? that contain the TME intact has shown promising responses to immunomodulators on native immune cells. We propose a microfluidic platform that enables drug treatment, exogenous T cell therapy, and high-content analysis using hundreds to thousands of similarly sized, precision-sliced cuboidal micro-tissues (CTs) produced from a single tumor sample. Here we propose a combination of two methodologies to demonstrate the feasibility of our approach: 1) precision slicing methodology that will produce large numbers of cuboidal micro-tissues (CTs) from a single tumor biopsy; and 2) microfluidic trapping of the CTs in a multi-well platform, allowing for drug application to each individual CT or groups of CTs. We will be able to obtain several hundred patient-derived CTs from each tumor resection. The size of the CTs (initially 400 m400 m400 m) will be reproducible and chosen to optimize viability and retention of the TME. As the CTs are cultured, their cuboidal shape will relax into a more rounded one. We will study the viability of the CTs and their TME composition as a function of size in various culture conditions, including collagen gels. We will focus on breast cancer immunotherapy using a syngeneic mouse breast tumor model. For this Aim, we will deliver various concentrations and combinations of immunomodulatory drugs, including antibody-based drugs, to breast tumor CTs in the microfluidic device, and examine the effects on the resident immune system. We will assess cytokine production and use high-content immunohistochemistry and bioinformatics analysis to assess immune cell engagement with different cell types as well as cell death. We will apply the platform to deliver immune checkpoint inhibitors (CTLA4, PD-L1, PD-1) and other immunomodulators (such as IL-10) and examine the effect on the immune state, cell death, and the behavior of resident T cells (activation and localization). In the R33 phase we plan on applying our microfluidic platform to CTs obtained from breast tumor patients (ongoing collaboration with Dr. V.K. Gadi, Fred Hutch).